EP0431905A1 - Method for purifying phage DNA - Google Patents
Method for purifying phage DNA Download PDFInfo
- Publication number
- EP0431905A1 EP0431905A1 EP90313173A EP90313173A EP0431905A1 EP 0431905 A1 EP0431905 A1 EP 0431905A1 EP 90313173 A EP90313173 A EP 90313173A EP 90313173 A EP90313173 A EP 90313173A EP 0431905 A1 EP0431905 A1 EP 0431905A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- phage
- proteins
- ultrafiltration
- phage dna
- membrane filter
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
Definitions
- the present invention relates to a method for purifying phage DNA.
- the method of the present invention may be used in the field of genetic engineering.
- nucleic acids are widely purified from various cells, viruses and phages.
- Purification of phage DNAs is generally carried out in this technique.
- the widely adopted conventional method for purifying phage DNAs from the culture medium comprises the steps of removing E . coli cells from the culture medium by centrifugation, precipitating the phage by treatment with polyethyleneglycol, removing proteins by phenol extraction and concentrating DNA by ethanol precipitation.
- the object of the present invention is to provide a process of purifying phage DNAs by which phage DNAs may be purified to high purity without centrifugation steps and without using reagents toxic to human.
- phage DNAs may be purified to high purity by removing E . coli cells by filtration through a membrane filter, decomposing and denaturing the phage proteins, and then removing the proteins by ultrafiltration, to complete the present invention.
- the present invention provides a method for purifying phage DNAs comprising, in the order mentioned, the steps of filtering a mixture containing microorganism cells and phage through a membrane filter to obtain a filtrate, so as to remove said microorganism cells; decomposing and denaturing phage proteins in said filtrate; and subjecting the resultant to ultrafiltration so as to remove impurities.
- phage DNAs may be purified to high purity with high yield from a culture medium without conducting a centrifugation step.
- the method since the centrifugation steps is not necessary, the method may be automatized. Further, with the method of the present invention, toxic reagents are not necessary and the time required for the purification is shortened.
- the drawing shows an agarose gel electrophoresis pattern of a phage DNA purified by the method of the present invention together with an electrophoresis pattern of the phage DNA purified by the conventional method and an electrophoresis pattern of a commercially available phage DNA.
- a mixture containing microorganism cells and phage is treated.
- Representative example of such a mixture is a culture medium of a microorganism which is infected with a virus.
- the culture medium may be treated as it is by the method of the present invention.
- a culture medium containing M13 phage and E . coli in 2 x TY medium or the like may be treated by the method of the present invention.
- the above-described mixture is filtered through a membrane filter so as to remove microorganism cells.
- the pore size of the membrane filter used here may preferably be 0.45 - 0.22 ⁇ m.
- the material constituting the membrane filter is not at all restricted.
- the phage proteins in the filtrate is decomposed and denatured.
- the decomposition and denaturation of the proteins may be carried out by treating the filtrate with a proteolytic enzyme such as Proteinase K.
- the concentration of the proteolytic enzyme may be appropriately selected.
- the concentration of the proteolytic enzyme may usually be 0.001 - 0.5 mg/ml.
- the decomposition and denaturation of the phage proteins may be carried out by treatment with an organic solvent, a surfactant or an alkali or by heat treatment or the like. More particularly, the step of decomposing and denaturing the proteins may be carried out by treating the protein with an organic solvent such as 30 - 100% methanol or ethanol.
- the separation of DNA and proteins may be attained by using a reagent which does not have a decomposing ability as strong as proteolytic enzymes or organic solvents, when a comparatively simple phage is to be treated.
- the decomposition and denaturation of proteins may be attained by treatment with a surfactant.
- the surfactant include anion surfactants such as sodium lauryl sulfate (SDS).
- SDS sodium lauryl sulfate
- the concentration thereof may preferably be 0.01 - 1% by weight.
- heat may be utilized for the protein-nucleic acid separation.
- the heat treatment may preferably be carried out at a temperature of 80 - 100°C for 5 - 20 minutes.
- the decomposition and denaturation of the proteins may also be carried out by alkali treatment.
- Preferred examples of the alkali include aqueous solution of alkaline metal hydroxides with a concentration of 0.1 - 1N.
- proteins can be removed from the phage DNA.
- the resulting solution is then ultrafiltered.
- the ultrafiltration membrane used herein preferably has a fractionation molecular weight of 20,000 to 1,000,000. If the molecular weight to be fractionated is smaller than this range, the efficiency of the removal of proteins is reduced. On the other hand, if the molecular weight is larger than this range, DNA may pass through the ultrafilter.
- the ultrafilter may be made of any material. For example, commercially available ultrafilters made of polysulfone may be employed. After the ultrafiltration, by washing the ultrafilter with an appropriate solution such as a buffer solution, the phage DNA may be recovered as a solution. To promote the yield of the phage DNA, it is preferred to wash the ultrafilter by passing the solution through the ultrafilter. The solution to be used for washing the ultrafilter is not restricted.
- TE-buffer Tris-HCl buffer containing EDTA
- TE-buffer Tris-HCl buffer containing EDTA
- the second step may be carried out immediately after the first step as described above.
- the obtained filtrate may be subjected to ultrafiltration to remove low molecular components, thereby purifying the phage.
- the ultrafiltration may be carried out in the same manner as in the third step just described above.
- the treatment for decomposing and denaturing the proteins in the second step may be carried out on the ultrafilter, and the ultrafiltration in the third step may be carried out on the same ultrafilter.
- E . coli JM109 was inoculated to 2 ⁇ TY medium, and then M13 phage was infected thereto. The medium was incubated at 37°C for 4 hours.
- the resulting culture medium was filtered through a membrane filter having a pore size of 0.45 ⁇ m under a pressure with nitrogen gas.
- the obtained filtrate was subjected to ultrafiltration through a ultrafilter having a fractionation molecular weight of 300,000 to remove low molecular weight components in the culture medium.
- a ultrafilter having a fractionation molecular weight of 300,000 to remove low molecular weight components in the culture medium.
- 5 ⁇ g/ ⁇ l solution of proteinase K was placed on the ultrafilter, and the reaction was allowed to occur for 10 minutes.
- the enzyme solution was removed under pressure, and the ultrafilter was washed with TE buffer by passing through the buffer under pressure through the ultrafilter. Two hundred microliters of TE buffer was placed on the ultrafilter and the ultrafilter was shaken. Thereafter, the TE buffer was recovered by using a pipette.
- phage DNA was subjected to agarose gel electrophoresis according to a conventional method.
- commercially available M13 phage DNA and the M13 phage DNA purified by the conventional purification method comprising polyethylene glycol precipitation and phenol extraction were also subjected to agarose gel electrophoresis.
- lane 1 shows an electrophoresis pattern of molecular weight markers consisting of Hin dIII digest of ⁇ phage
- lane 2 shows an electrophoresis pattern of commercially available M13 phage single-stranded DNA
- lane 3 shows an electrophoresis pattern of M13 phage DNA purified by the conventional polyethylene glycol precipitation and phenol extraction
- lane 4 shows an electrophoresis pattern of M13 phage DNA purified by the method of the present invention.
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- Crystallography & Structural Chemistry (AREA)
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Abstract
Description
- The present invention relates to a method for purifying phage DNA. The method of the present invention may be used in the field of genetic engineering.
- Recently, with the progress of the genetic engineering techniques, nucleic acids are widely purified from various cells, viruses and phages. For determining base sequences of DNAs carrying genetic information, it is an important technique to replicate single-stranded DNAs by culturing M13 phage with E. coli. Purification of phage DNAs is generally carried out in this technique. The widely adopted conventional method for purifying phage DNAs from the culture medium comprises the steps of removing E. coli cells from the culture medium by centrifugation, precipitating the phage by treatment with polyethyleneglycol, removing proteins by phenol extraction and concentrating DNA by ethanol precipitation.
- However, with this conventional method, reagents which are toxic to human, such as phenol and chloroform must be used. Further, since centrifugation is necessary in each step, it is difficult to automatize the method. For automatizing the purification, a method which does not require centrifugation is desired.
- Accordingly, the object of the present invention is to provide a process of purifying phage DNAs by which phage DNAs may be purified to high purity without centrifugation steps and without using reagents toxic to human.
- The present inventors intensively studied to find that phage DNAs may be purified to high purity by removing E. coli cells by filtration through a membrane filter, decomposing and denaturing the phage proteins, and then removing the proteins by ultrafiltration, to complete the present invention.
- That is, the present invention provides a method for purifying phage DNAs comprising, in the order mentioned, the steps of filtering a mixture containing microorganism cells and phage through a membrane filter to obtain a filtrate, so as to remove said microorganism cells; decomposing and denaturing phage proteins in said filtrate; and subjecting the resultant to ultrafiltration so as to remove impurities.
- By the method of the present invention, phage DNAs may be purified to high purity with high yield from a culture medium without conducting a centrifugation step. According to the method of the present invention, since the centrifugation steps is not necessary, the method may be automatized. Further, with the method of the present invention, toxic reagents are not necessary and the time required for the purification is shortened.
- The drawing shows an agarose gel electrophoresis pattern of a phage DNA purified by the method of the present invention together with an electrophoresis pattern of the phage DNA purified by the conventional method and an electrophoresis pattern of a commercially available phage DNA.
- In the method of the present invention, a mixture containing microorganism cells and phage is treated. Representative example of such a mixture is a culture medium of a microorganism which is infected with a virus. The culture medium may be treated as it is by the method of the present invention. For example, a culture medium containing M13 phage and E. coli in 2 x TY medium or the like may be treated by the method of the present invention.
- In the first step of the method of the present invention, the above-described mixture is filtered through a membrane filter so as to remove microorganism cells. The pore size of the membrane filter used here may preferably be 0.45 - 0.22 µm. The material constituting the membrane filter is not at all restricted.
- In the second step, the phage proteins in the filtrate is decomposed and denatured. The decomposition and denaturation of the proteins may be carried out by treating the filtrate with a proteolytic enzyme such as Proteinase K. In this case, the concentration of the proteolytic enzyme may be appropriately selected. When a culture medium containing M13 phage and E. coli is treated, the concentration of the proteolytic enzyme may usually be 0.001 - 0.5 mg/ml. As described later, it is preferred to subject the filtrate to ultrafiltration before the treatment with a proteolytic enzyme and to add the proteolytic enzyme to the phage remained on the ultrafiltration membrane, although this ultrafiltration step is not required.
- Other than the treatment with a proteolytic enzyme, the decomposition and denaturation of the phage proteins may be carried out by treatment with an organic solvent, a surfactant or an alkali or by heat treatment or the like. More particularly, the step of decomposing and denaturing the proteins may be carried out by treating the protein with an organic solvent such as 30 - 100% methanol or ethanol. The separation of DNA and proteins may be attained by using a reagent which does not have a decomposing ability as strong as proteolytic enzymes or organic solvents, when a comparatively simple phage is to be treated. For example, the decomposition and denaturation of proteins may be attained by treatment with a surfactant. Preferred examples of the surfactant include anion surfactants such as sodium lauryl sulfate (SDS). In case of using SDS, the concentration thereof may preferably be 0.01 - 1% by weight. Since proteins are weak to heat and nucleic acids such as DNAs are relatively strong to heat, heat may be utilized for the protein-nucleic acid separation. In this case, the heat treatment may preferably be carried out at a temperature of 80 - 100°C for 5 - 20 minutes. The decomposition and denaturation of the proteins may also be carried out by alkali treatment. Preferred examples of the alkali include aqueous solution of alkaline metal hydroxides with a concentration of 0.1 - 1N.
- The above-mentioned treatments may be carried out individually or in combination. By this step, proteins can be removed from the phage DNA.
- The resulting solution is then ultrafiltered. The ultrafiltration membrane used herein preferably has a fractionation molecular weight of 20,000 to 1,000,000. If the molecular weight to be fractionated is smaller than this range, the efficiency of the removal of proteins is reduced. On the other hand, if the molecular weight is larger than this range, DNA may pass through the ultrafilter. The ultrafilter may be made of any material. For example, commercially available ultrafilters made of polysulfone may be employed. After the ultrafiltration, by washing the ultrafilter with an appropriate solution such as a buffer solution, the phage DNA may be recovered as a solution. To promote the yield of the phage DNA, it is preferred to wash the ultrafilter by passing the solution through the ultrafilter. The solution to be used for washing the ultrafilter is not restricted. Widely used buffer solutions such as TE-buffer (Tris-HCl buffer containing EDTA) may be employed as the solution for washing the ultrafilter. By this ultrafiltration step, the proteins and other impurities are removed from the solution obtained in the second step, so that phage DNA is purified.
- The second step may be carried out immediately after the first step as described above. However, after the first step, the obtained filtrate may be subjected to ultrafiltration to remove low molecular components, thereby purifying the phage. In this case, the ultrafiltration may be carried out in the same manner as in the third step just described above. Further, if this ultrafiltration step is employed, the treatment for decomposing and denaturing the proteins in the second step may be carried out on the ultrafilter, and the ultrafiltration in the third step may be carried out on the same ultrafilter.
- The invention will now be described by way of an example thereof. The example is presented for illustration purpose only and should not be interpreted in any restrictive way.
- E. coli JM109 was inoculated to 2 × TY medium, and then M13 phage was infected thereto. The medium was incubated at 37°C for 4 hours.
- The resulting culture medium was filtered through a membrane filter having a pore size of 0.45 µm under a pressure with nitrogen gas.
- The obtained filtrate was subjected to ultrafiltration through a ultrafilter having a fractionation molecular weight of 300,000 to remove low molecular weight components in the culture medium. To decompose the proteins of the phage, 5 µg/µl solution of proteinase K was placed on the ultrafilter, and the reaction was allowed to occur for 10 minutes.
- The enzyme solution was removed under pressure, and the ultrafilter was washed with TE buffer by passing through the buffer under pressure through the ultrafilter. Two hundred microliters of TE buffer was placed on the ultrafilter and the ultrafilter was shaken. Thereafter, the TE buffer was recovered by using a pipette.
- By this procedure, 2.5 µg of phage DNA was recovered from 2 ml of the culture medium.
- The thus obtained phage DNA was subjected to agarose gel electrophoresis according to a conventional method. As controls, commercially available M13 phage DNA and the M13 phage DNA purified by the conventional purification method comprising polyethylene glycol precipitation and phenol extraction were also subjected to agarose gel electrophoresis.
- The results are shown in the drawing. In the drawing,
lane 1 shows an electrophoresis pattern of molecular weight markers consisting of HindIII digest of λ phage,lane 2 shows an electrophoresis pattern of commercially available M13 phage single-stranded DNA,lane 3 shows an electrophoresis pattern of M13 phage DNA purified by the conventional polyethylene glycol precipitation and phenol extraction, andlane 4 shows an electrophoresis pattern of M13 phage DNA purified by the method of the present invention. - As is apparent from the drawing, it was confirmed that the phage DNA was purified to high purity by the method of the present invention.
- Although the present invention was described by way of preferred embodiment thereof, it is apparent for those skilled in the art that various modifications may be made without departing from the spirit and scope of the present invention.
Claims (5)
- A method for purifying phage DNA comprising, in the order mentioned, the steps of:
filtering a mixture containing microorganism cells and phage through a membrane filter to obtain a filtrate, so as to remove the said microorganism cells;
decomposing and denaturing phage proteins in the said filtrate; and
subjecting the resultant to ultrafiltration so as to remove impurities. - A method as claimed in Claim 1, characterised in that the pore size of the said membrane filter is 0.45 µm to 0.22 µm.
- A method as claimed in Claim 1 or Claim 2 characterised in that the molecular weight to be fractionated by the said ultrafiltration is in the range 20,000 to 1,000,000.
- A method as claimed in Claim 1, 2 or 3 characterised in that the step of decomposing and denaturing phage proteins is carried out by treating the said phage proteins with a proteolytic enzyme.
- A method as claimed in Claim 1, 2, 3 or 4 characterised in that it further comprises the step of ultrafiltration of the said filtrate obtained in the first step from the membrane filter before the said step of decomposing and denaturing the phage proteins.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP1317114A JP2978518B2 (en) | 1989-12-06 | 1989-12-06 | Method for purifying phage DNA |
JP317114/89 | 1989-12-06 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0431905A1 true EP0431905A1 (en) | 1991-06-12 |
EP0431905B1 EP0431905B1 (en) | 1994-05-11 |
Family
ID=18084596
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19900313173 Expired - Lifetime EP0431905B1 (en) | 1989-12-06 | 1990-12-05 | Method for purifying phage DNA |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP0431905B1 (en) |
JP (1) | JP2978518B2 (en) |
DE (1) | DE69008825T2 (en) |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0517515A2 (en) * | 1991-06-04 | 1992-12-09 | Tosoh Corporation | Method of purifying DNA |
WO1993001312A1 (en) * | 1991-07-09 | 1993-01-21 | Bertin & Cie | Cartridge, device and method for preparing purified nucleic acids from a cell sample |
WO1993011218A1 (en) * | 1991-12-02 | 1993-06-10 | Qiagen Gmbh | Method and device for the isolation of cell components, such as nucleic acids, from natural sources |
FR2712511A1 (en) * | 1993-11-16 | 1995-05-24 | Bertin & Cie | Cartridge for the preparation of purified nucleic acids. |
US6011148A (en) * | 1996-08-01 | 2000-01-04 | Megabios Corporation | Methods for purifying nucleic acids |
WO2000005358A1 (en) * | 1998-07-23 | 2000-02-03 | Valentis, Inc. | Methods for purifying nucleic acids |
WO2000066723A1 (en) * | 1999-05-04 | 2000-11-09 | Millipore Corporation | Method of ultrafiltration |
US6498240B1 (en) | 1999-09-17 | 2002-12-24 | Millipore Corporation | Method for sequencing reaction cleanup by constant pressure diffential ultrafiltration |
US7026468B2 (en) | 1996-07-19 | 2006-04-11 | Valentis, Inc. | Process and equipment for plasmid purification |
US7314746B2 (en) | 2002-09-13 | 2008-01-01 | Valentis, Inc. | Apparatus and method for preparative scale purification of nucleic acids |
US8008475B1 (en) * | 1997-10-23 | 2011-08-30 | Qiagen Gmbh | Method for isolating and purifying nucleic acids on surfaces |
CN115354041A (en) * | 2022-10-19 | 2022-11-18 | 北京君全智药生物科技有限公司 | Preparation method of M13 phage single-stranded DNA |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE102008055606A1 (en) | 2008-11-03 | 2010-05-06 | Bayer Technology Services Gmbh | A method for the high-throughput preparation of sequencing-competent DNA from individual plaques of peptides presenting phages |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0240191A2 (en) * | 1986-03-13 | 1987-10-07 | Seiko Instruments Inc. | Method of isolating DNA contained in a virus or cell |
WO1989001035A1 (en) * | 1987-07-23 | 1989-02-09 | Europäisches Laboratorium Für Molekularbiologie (E | Process and device for purification of m-13 phage dna |
EP0376080A1 (en) * | 1988-12-22 | 1990-07-04 | Talent SRL | Method for the extraction and purification of DNA |
-
1989
- 1989-12-06 JP JP1317114A patent/JP2978518B2/en not_active Expired - Fee Related
-
1990
- 1990-12-05 DE DE1990608825 patent/DE69008825T2/en not_active Expired - Fee Related
- 1990-12-05 EP EP19900313173 patent/EP0431905B1/en not_active Expired - Lifetime
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0240191A2 (en) * | 1986-03-13 | 1987-10-07 | Seiko Instruments Inc. | Method of isolating DNA contained in a virus or cell |
WO1989001035A1 (en) * | 1987-07-23 | 1989-02-09 | Europäisches Laboratorium Für Molekularbiologie (E | Process and device for purification of m-13 phage dna |
EP0376080A1 (en) * | 1988-12-22 | 1990-07-04 | Talent SRL | Method for the extraction and purification of DNA |
Cited By (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0517515A3 (en) * | 1991-06-04 | 1993-06-16 | Tosoh Corporation | Method of purifying dna |
EP0517515A2 (en) * | 1991-06-04 | 1992-12-09 | Tosoh Corporation | Method of purifying DNA |
WO1993001312A1 (en) * | 1991-07-09 | 1993-01-21 | Bertin & Cie | Cartridge, device and method for preparing purified nucleic acids from a cell sample |
US5352609A (en) * | 1991-07-09 | 1994-10-04 | Bertin & Cie | Cartridge, apparatus, and method for preparing purified nucleic acids from a cell sample |
US6277648B1 (en) | 1991-12-02 | 2001-08-21 | Qiagen Gmbh | Process and a device for the isolation of cell components such as nucleic acids from natural sources |
WO1993011218A1 (en) * | 1991-12-02 | 1993-06-10 | Qiagen Gmbh | Method and device for the isolation of cell components, such as nucleic acids, from natural sources |
US6609618B2 (en) | 1991-12-02 | 2003-08-26 | Qiagen Gmbh | Device for the isolation of cell components such as nucleic acids from natural sources |
FR2712511A1 (en) * | 1993-11-16 | 1995-05-24 | Bertin & Cie | Cartridge for the preparation of purified nucleic acids. |
WO1995014086A1 (en) * | 1993-11-16 | 1995-05-26 | Bertin & Cie | Cartridge for preparing purified nucleic acids |
US5789243A (en) * | 1993-11-16 | 1998-08-04 | Bertin & Cie | Cartridge for preparing purified nucleic acids |
US7026468B2 (en) | 1996-07-19 | 2006-04-11 | Valentis, Inc. | Process and equipment for plasmid purification |
US8236495B2 (en) | 1996-07-19 | 2012-08-07 | Samuel Nochumson | Process and equipment for plasmid purification |
US6011148A (en) * | 1996-08-01 | 2000-01-04 | Megabios Corporation | Methods for purifying nucleic acids |
US7807822B2 (en) | 1996-08-01 | 2010-10-05 | Robert Bridenbaugh | Methods for purifying nucleic acids |
US8008475B1 (en) * | 1997-10-23 | 2011-08-30 | Qiagen Gmbh | Method for isolating and purifying nucleic acids on surfaces |
WO2000005358A1 (en) * | 1998-07-23 | 2000-02-03 | Valentis, Inc. | Methods for purifying nucleic acids |
WO2000066723A1 (en) * | 1999-05-04 | 2000-11-09 | Millipore Corporation | Method of ultrafiltration |
US6498240B1 (en) | 1999-09-17 | 2002-12-24 | Millipore Corporation | Method for sequencing reaction cleanup by constant pressure diffential ultrafiltration |
US6645722B2 (en) | 1999-09-17 | 2003-11-11 | Millipore Corporation | Method for sequencing reaction cleanup by constant differential pressure ultrafiltration |
US7314746B2 (en) | 2002-09-13 | 2008-01-01 | Valentis, Inc. | Apparatus and method for preparative scale purification of nucleic acids |
US7771945B2 (en) | 2002-09-13 | 2010-08-10 | Au-Yeung Kwok-Leung Jacky | Apparatus and method for preparative scale purification of nucleic acids |
CN115354041A (en) * | 2022-10-19 | 2022-11-18 | 北京君全智药生物科技有限公司 | Preparation method of M13 phage single-stranded DNA |
Also Published As
Publication number | Publication date |
---|---|
JPH03180182A (en) | 1991-08-06 |
EP0431905B1 (en) | 1994-05-11 |
DE69008825D1 (en) | 1994-06-16 |
DE69008825T2 (en) | 1994-08-25 |
JP2978518B2 (en) | 1999-11-15 |
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